The objective of this project is to identify cellular targets of the smallpox D4R virulence factor, with the ultimate goal of identifying pharmacological inhibitors specific for the viral protein while minimizing deleterious effects on the homologous cellular proteins. We have identified and characterized the two primary cellular genes of the makorin (MKRN) gene family that encode proteins of unknown function containing multiple zinc-finger motifs. We also provide evidence that the poxvirulence factor, D4R, is derived from a transduced ancestral MKRN cDNA. D4R orthologs are frequently mutated in attenuated vaccinia viruses, and deletion of the mousepox orthologous p28 gene renders the virus non-lethal. Published and anecdotal evidence support a model in which D4R intervenes in a makorin pathway to prevent normal antiviral apoptotic culling. The molecular mechanism is likely to involve the conserved RING zinc-finger in the cellular and viral derivatives to function as an E3 ubiquitin ligase, targeting specific apoptotic regulators for degradation. It is anticipated that the cellular and viral proteins will have shared activity and specificity profiles. In this grant proposal, we would like to directly address these issues using a combination of mouse models and protein analyses. Our specific aims are: (1) to evaluate apoptotic roles for makorin-1, makorin-2, and D4R proteins in mouse models; (2) to determine molecular targets of makorin and D4R proteins; and (3) to assess whether the interacting targets are ubiquitinated by makorin and D4R proteins. Mouse models created in this proposal will be used for future in vivo studies of ectromelia virulence, and the identification of molecular targets of D4R/makorin proteins will provide insight into this important poxvirus/host cell interaction.